Biosensor

ABSTRACT

To provide an improved biosensor which allows easy addition of whole blood to the sensor and rapid supply of the added whole blood to a filter even in the case of collecting blood by fingertip centesis for measurement, a second air aperture is provided in a sample solution supply pathway including an electrode system and a reaction layer and communicating with a first air aperture on the terminal end side.

TECHNICAL FIELD

The present invention relates to a biosensor, specifically a cholesterolsensor, capable of determining the quantity of a specific component in asample in a speedy, highly-sensitive and simple manner.

BACKGROUND ART

A description is given to an example of a conventional biosensor interms of a glucose sensor.

A typical glucose sensor is obtained by forming an electrode systemincluding at least a measuring electrode and a counter electrode on aninsulating base plate by screen printing or the like and forming anenzyme reaction layer containing a hydrophilic polymer, oxidoreductaseand an electron mediator on the electrode system. For example, glucoseoxidase is used as oxidoreductase and a metal complex or an organiccompound such as potassium ferricyanide, ferrocene derivatives andquinone derivatives is used as the electron mediator. A buffer is addedto the enzyme reaction layer if required.

Upon dropping a sample solution containing a substrate onto the enzymereaction layer of the biosensor, the enzyme reaction layer dissolves tocause a reaction between the enzyme and the substrate. Reduction of theelectron mediator accompanies the reaction. After the enzyme reaction iscompleted, the reduced electron mediator is electrochemically oxidizedto obtain an oxidation current value, from which the substrateconcentration in the sample solution is determined.

The biosensor of this kind is theoretically capable of measuring varioussubstances by using an enzyme of which the substrate is a measuringobject. For example, if cholesterol oxidase or cholesterol dehydrogenaseis used as oxidoreductase, a cholesterol value in serum, which is usedas a diagnostic index in various medical institutions, can be measured.

In this case, the enzyme reaction of cholesterol esterase proceeds veryslowly. Accordingly, an appropriate surfactant may be added to improvethe activity of cholesterol esterase and reduce the time required forthe whole reaction. However, the surfactant included in the reactionsystem affects adversely on hemocytes, which makes impossible to measurewhole blood as done in the glucose sensor.

In response to this, a proposal has been made to provide a filter(hemocyte filtering part) in the vicinity of an opening of a samplesolution supply pathway so that plasma obtained by filtering hemocytesout of the whole blood is exclusively and rapidly supplied into a sensor(sample solution supply pathway).

However, if the filter is inappropriately built in the sensor, hemocytescaptured in the filter are destroyed to dissolve hemoglobin out.Thereby, filtration of the hemocyte components with the filter becomesdifficult and small hemoglobin flows into the sample solution supplypathway to cause a measurement error.

This is presumably caused by the fact that a difference in thicknessbetween the filter before absorbing a sample solution and the filterexpanded after absorbing the sample solution is not fitted with a gapbetween pressing parts for holding the filter from the top and thebottom. When the gap between the pressing parts for holding the filterfrom the top and the bottom is too narrow for the thickness of theexpanded filter, the expansion of the filter is prevented. The pore sizeof the filter thus prevented from expansion cannot be widenedsufficiently, thereby the hemocytes as infiltrating thereinto aredestroyed.

As opposed to this, if the gap between the upper and lower pressingparts is previously set wide for the supposed thickness of the expandedfilter taking into account that the degree of the filter expansionvaries depending on a hematocrit value (volume percent of red cell)different in each sample solution, it is feared that the filter may bemisaligned during storage of the sensor.

It is also considered that the filter itself is made thinner than aconventional one to prevent the filter from expansion due to theabsorption of the sample solution. In this case, however, if the samplesolution is sucked only from an end of the filter on a primary side, theamount of the sample solution absorbed within a certain period of timeis reduced as described in the specification of Japanese PatentApplication No. 2000-399056. Then, the rate at which the plasma flowsout of a secondary-side of the filter is reduced and the rate at whichthe plasma saturates the inside of the sensor, in particular the insideof the sample solution supply pathway, becomes low, which results inlong measurement time.

As opposed to this, where a suction area is made wider to increase theamount of the sample solution that can be absorbed within a certainperiod of time and the sample solution is dropped on an upper part ofthe filter, the sample solution flows along the surface of the filter ata higher rate than the rate of infiltration into the filter. The samplesolution having flown along the filter surface then flows into thesample solution supply pathway from an opening thereof connecting thesample solution supply pathway and the filter, which may cause ameasurement error.

In the specification of Japanese Patent Application No. 2001-152868, forexample, there is disclosed a technique of providing a first pressingpart for holding a primary side portion of the filter from the bottom,second pressing parts for holding a secondary side portion of the filterfrom the top and the bottom, a third pressing part for holding a centerportion of the filter from the top and a void provided between thesecond and third pressing parts for surrounding the filter. With thistechnique, the destruction of hemocytes caused by the prevention of thefilter expansion is inhibited even if the gap between the pressing partsfor holding the filter from the top and the bottom is not fitted withthe thickness of the expanded filter. It is also described that themeasurement error caused by hemocytes flown into the sample solutionsupply pathway along the filter surface is avoided by dropping thesample solution directly onto the filter.

In the case of a sensor to which the filter is applied, however, theplasma may spill from an air aperture in some cases depending on theviscosity of the plasma or the plasma amount in whole blood after thefiltered plasma flows into and saturates the sample solution supplypathway, though the plasma is expected to stop at the air aperture. Thewhole blood, which is the sample solution, comprises hemocyte componentsand a liquid component (plasma), in which the percentage of the hemocytecomponents (volume percent of red cell) is in the range of about 20 to60% though it varies among individuals. Further, the viscosity variesdepending on the cholesterol concentration in the whole blood. Thesedifferences cause a problem in that the plasma travels along theelectrode plate to spill from the sample solution supply pathway afterreaching the air aperture if the sample solution, even of the sameorigin, has different flow rate or reduced viscosity and volume percentin red cell.

Hence, the present invention is intended to provide a biosensor that isimproved to eliminate the above-described disadvantages and stop theplasma at the air aperture irrespective of the viscosity of the plasmain the sample solution and the plasma amount in the whole blood.Further, the present invention is intended to provide a cholesterolsensor for measuring whole blood with high accuracy and excellentresponse.

DISCLOSURE OF INVENTION

The present invention relates to a biosensor comprising an insulatingbase plate, an electrode system including a measuring electrode and acounter electrode provided on the base plate, a cover for covering theinsulating base plate, at least one reaction layer containingoxidoreductase and/or an electron mediator, a sample solution supplypathway which is formed by the insulating base plate and the cover andincludes the electrode system and the reaction layer, a first airaperture provided in the cover in the sample solution supply pathway, asample solution supply part and a filter provided between the samplesolution supply pathway and the sample solution supply part to filterhemocytes, the biosensor being capable of sucking plasma with hemocytestherein filtered with the filter into the sample solution supply pathwaydue to capillarity,

characterized in that a second air aperture is provided in theinsulating base plate in the sample solution supply pathway.

In the above biosensor, it is preferred that the first air aperture andthe second air aperture are communicated.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a perspective view of a disassembled biosensor according to anembodiment of the present invention.

FIG. 2 is a perspective view of an assembled biosensor according to anembodiment of the present invention.

FIG. 3 is a schematic vertical section of the sensor excluding areaction layer and the like.

FIG. 4 is a schematic vertical section illustrating the vicinity of anelectrode system of the sensor.

FIG. 5 is a graph illustrating a response characteristic of acholesterol sensor according to an example of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

As described above, the biosensor according to the present inventionincludes an electrode system and a reaction layer and has a filter forfiltering hemocytes provided between a sample solution supply pathwayhaving a first air aperture on a terminal end side and a sample solutionsupply part. Plasma from which hemocytes are removed by the filter issucked into the sample solution supply pathway due to capillarity. Thebiosensor is characterized in that the sample solution supply pathwayincludes a second air aperture in addition to the first air aperture.

Further, the first air aperture is preferably communicated with thesecond air aperture.

With the above-described structure, the plasma can be kept in the samplesolution supply pathway regardless of differences in viscosity of theplasma in the sample solution, plasma amount in whole blood and volumepercent of red cell. Conventionally, the plasma flown in the pathwaytravels along the insulating base plate to spill from the samplesolution supply pathway together with a reagent upon reaching the airaperture, thereby causing a measurement error. However, with the secondair aperture opened in the insulating base plate to communicate with thefirst air aperture, the sample solution is prevented from travelingalong the insulating base plate and spilling from the sample solutionsupply pathway. Thereby, the volume of a filtrate to be measured isautomatically determined irrespective of the viscosity of the samplesolution and the percentage of sample components to be filtered. Thismakes possible to resolve the measurement error. Further, since thesample solution exists only in the sample solution supply pathway, thepossibility of contamination of the other parts than the sensor(measurement devices and a measurer) is reduced.

The electron mediator used in the present invention may be selectedfrom, besides potassium ferricyanide, redox compounds having electrontransferring ability to and from oxidoreductase such as cholesteroloxidase.

Oxidoreductase is an enzyme of which the substrate is a measuringobject. In a sensor whose measuring object is glucose, glucose oxidaseis used. In order to measure a cholesterol value in serum, which is usedas a diagnostic index, are used cholesterol oxidase or cholesteroldehydrogenase which is an enzyme for catalyzing oxidative reaction ofcholesterol and cholesterol esterase which is an enzyme for catalyzing aprocess of converting cholesterol ester into cholesterol. Since theenzyme reaction of cholesterol esterase proceeds very slowly, anappropriate surfactant may be added, for example, to improve theactivity of cholesterol esterase and reduce the time required for thewhole reaction.

A layer containing the electron mediator and a reaction layer containingoxidoreductase are arranged on or in the vicinity of the electrodesystem in the sensor. In a sensor including a covering member, which iscombined with the base plate having the electrode system providedthereon to form therebetween a sample solution supply pathway forsupplying a sample solution to the electrode system, the reaction layermay be arranged on a portion exposed to the sample solution supplypathway or an opening of the sample solution supply pathway. In eitherposition, it is preferred that the reaction layer is easily dissolved bythe introduced sample solution to reach the electrode system.

For the purpose of protecting the electrodes and inhibiting the reactionlayer to be formed from peeling, a hydrophilic polymer layer ispreferably formed on the electrode system. Other than on the electrodesystem, the hydrophilic polymer layer is preferably formed as a base forforming the reaction layer or a hydrophilic polymer may be contained inthe reaction layer lying at the bottom.

Above all, it is preferable that the reaction layer containing theelectron mediator is separated from the surfactant to enhance thesolubility of the reaction layer. It is also preferable that theelectron mediator is separated from cholesterol oxidase and cholesterolesterase, which are enzymes for catalyzing the oxidative reaction ofcholesterol, in view of stability during storage.

There is an example of a biosensor for measuring blood sugar level inwhich a lipid-containing layer is formed to cover the layers formed onthe electrode system such that the sample solution is introduced to thereaction layer (for example, Japanese Laid-Open Patent Publication No.HEI 2-0062952). In the biosensor according to the present invention formeasuring cholesterol, it is preferred that part of the reaction layeris formed by a freeze drying method (e.g., the specification of JapanesePatent Application No. 2000-018834) or the surface of the coveringmember is given hydrophilicity by means of a surfactant or plasmairradiation. Such a structure can eliminate the need of forming thelipid layer.

As the hydrophilic polymer, for example, may be used water-solublecellulose derivatives, in particular ethyl cellulose, hydroxypropylcellulose, carboxymethyl cellulose, polyvinylpyrrolidone, polyvinylalcohol, gelatin, agarose, polyacrylic acid or salts thereof, starch orderivatives thereof, polymers of maleic anhydride or salts thereof,polyacrylamide, methacrylate resin and poly-2-hydroxyethyl methacrylate.

As the surfactant, for example, may be used n-octyl-β-D-thioglucoside,polyethylene glycol monododecyl ether, sodium cholate,dodecyl-β-maltoside, sucrose monolaurate, sodium deoxycholate, sodiumtaurodeoxycholate, N,N-bis(3-D-gluconamidopropyl)deoxycholamide andpolyoxyethylene (10) octyl phenyl ether.

In the case of using lipid, amphipathic phospholipid such as lecithin,phosphatidyl choline and phosphatidylethanolamine is favorably used.

An oxidation current may be measured by a measurement method on atwo-electrode system using only a measuring electrode and a counterelectrode or a three-electrode system using a reference electrode inaddition, among which the three-electrode system allows measurement withgreater accuracy.

Hereinafter, the present invention will be detailed by way of specificembodiments with reference to the figures. FIG. 1 is a perspective viewof a disassembled biosensor according to a preferred embodiment of thepresent invention.

In the biosensor shown in FIG. 1, an electrode system including aworking electrode 2 and a counter electrode 3 is formed by sputteringusing palladium and subsequent laser trimming on the left side of aninsulating base plate 1 made of insulating resin such as polyethyleneterephthalate. An area of the electrodes is determined in correspondencewith a width of a slit 12 formed in a spacer 7 to be described later.The insulating base plate 1 also includes an adhesive part 4 and anaperture 5. The adhesive part 4 may be provided by applying, forexample, a double-stick tape on the insulating base plate 1.

The spacer 7 is provided with an opening 10 for accommodating a filter 6therein, a slit 12 for forming a sample solution supply pathway 12′,rails 9 formed on both sides of the slit 12 to introduce a samplesolution into a primary side portion of the filter and a connecting part11 for connecting the opening 10 and the slit 12.

A cover 13 includes a first air aperture 17, an opening 16 and rails 15formed on both sides of the opening 16 to introduce the sample solutioninto the primary side portion of the filter.

A spacer 18 includes an opening 21 for accommodating the filter 6therein and rails 20 formed on both sides of the opening 21 to introducethe sample solution into the primary side portion of the filter.

A cover 22 includes an opening 25 for accommodating the filter 6therein, a pressing part (dividing part) 26, an aperture 27 and rails 24formed on both sides of the opening 25 to introduce the sample solutioninto the primary side portion of the filter.

Upon integrating the members shown in FIG. 1, the opening 10 in thespacer 7, the opening 16 in the cover 13, the opening 21 in the spacer18 and the opening 25 in the cover 22 shown in FIG. 1 are communicated.Further, a second air aperture 31 in the insulating base plate 1, aterminal end of the slit 12 in the spacer 7 and the first air aperture17 in the cover 13 are communicated.

The filter 6 is made of glass fiber filter paper and has an isoscelestriangle shape as viewed in a projection on a plane identical to theinsulating base plate 1 shown in FIG. 1.

In assembling the sensor, first, the cover 13 is placed on the spacer 7in a positional relationship as indicated by dashed lines in FIG. 1 toobtain a joint base plate A. At this time, the slit 12 forms a concaveportion in the cover 13 and the spacer 7 thus jointed, in which areaction layer is formed as described later.

Then, the cover 22 is placed on the spacer 18 in a positionalrelationship as indicated by dashed lines in FIG. 1 to obtain a jointbase plate B.

Further, the insulating base plate 1 and the joint base plates A and Bare assembled in a positional relationship as indicated by dashed linesin FIG. 1 and the filter 6 is mounted thereon in such a manner that thefilter 6 having an almost isosceles triangle shape in a projection on aplane identical to the insulating base plate 1 contacts the adhesivepart 4 of the insulating base plate 1 at the right end on the primaryside (bottom side).

In other words, the right end on the primary side (bottom side) of thefilter 6 enters a state of being disposed on the insulating base plate 1and fitted into the opening 10 of the spacer 7, the opening 16 of thecover 13, the opening 21 of the spacer 18 and the opening 25 of thecover 22. The left end on a secondary side (vertex side) of the filter 6is brought into a state of being sandwiched between the connecting part11 in the concave portion of the joint base plate A and the insulatingbase plate 1.

FIG. 2 shows a schematic perspective view of the thus obtained biosensoraccording to the present invention and FIG. 3 shows its structure insection. FIG. 3 is a schematic vertical section of the biosensoraccording to the present invention taken along the line X—X shown inFIG. 2. In FIG. 3, reaction layers and the like provided in the samplesolution supply pathway 12′ are omitted.

In the biosensor of the present invention shown in FIGS. 1 to 3,apertures 5 and 27 are formed as shown in FIG. 3, in which the filter 6is not in contact with the other members.

That is, the biosensor of the present invention includes, as shown inFIG. 3, a first pressing part a for holding the primary side portion ofthe filter 6 from the bottom, second pressing parts b and b′ for holdingthe secondary side portion of the filter 6 from the top and the bottomand a third pressing part c for holding the center of the filter 6 fromthe top.

Between the second pressing parts b and b′ and the third pressing partc, the apertures 5 and 27 are communicated via the openings 10, 16 and21 (see FIG. 1) to make the filter 6 not contact the other members.

Further, opening ends 8, 14, 19 and 23 shown in FIG. 1 are alsocommunicated to form a concave portion which serves as a sample solutionsupply part 30 as shown in FIG. 2. In the biosensor of the presentinvention, the existence of the concave portion makes an end face of thesensor open (open to the outside). Therefore, as a method of adding thesample solution, for example, a fingertip stung to bleed may be rubbedagainst the sample solution supply part 30. The sample solution istemporarily held in the concave portion and then supplied rapidly andintensively to the primary side of the filter.

FIG. 4 shows a schematic vertical section illustrating anotherembodiment of the biosensor of the present invention. The reactionlayers and the electrode system omitted in FIG. 3 are depicted in FIG.4. On the electrode system (2 and 3) of the insulating base plate 1, ahydrophilic polymer layer 28 and a reaction layer 29 are formed. Inaddition, a reaction layer 29′ is formed on the underside of the cover13 corresponding to a ceiling of the sample solution supply pathway 12′.Other members shown in FIG. 4 are the same as those shown in FIG. 3.

The biosensor shown in FIGS. 1 to 4 is made of six members including thefilter and various base plates for easy explanation of the structure.However, the cover 22 and the spacer 18 or the cover 13 and the spacer 7may be formed as a single member.

In measuring cholesterol in blood with this sensor, whole blood issupplied from the sample solution supply part 30, the concave portion,to the filter 6. At this time, since the spacer 7, the cover 13, thespacer 18 and the cover 22 contact the sample solution supply part 30 atthe rails 9, 15, 20 and 24, the whole blood is efficiently supplied tothe filter 6.

The supplied blood permeates into the filter 6 from the end face and thetop face on the primary side. In the filter 6, since the permeation rateof hemocytes is lower than that of plasma which is a liquid component,the plasma seeps from the tip of the filter 6 on the secondary side. Theseeped plasma fills the vicinity of the electrode system and the entiresample solution supply pathway 12′ extending to the connecting partbetween the first and second air apertures 17 and 31 while dissolvingthe reaction layer carried on a position covering the electrode systemand/or the underside of the cover 13.

Once the entire sample solution supply pathway 12′ is filled up, theliquid flow in the filter 6 stops. At this time, the hemocytes remain inthe filter 6 without reaching the secondary side end of the filter 6. Itis therefore necessary to design the filter 6 to give a difference inflow resistance between the plasma and the hemocytes to such an extentthat the hemocytes do not reach the secondary side end of the filter 6even if the plasma are passed in an amount enough to fill the samplesolution supply pathway 12′.

A depth filter having a pore diameter of 1 to 7 μm is suitably used asthe filter of the present invention. The thickness of the filter ispreferably 300 to 400 μm.

Through the process of hemocyte filtration, a chemical reaction occursbetween the reaction layer dissolved by the plasma and a component to bemeasured in the plasma (cholesterol in using a cholesterol sensor).After an elapse of a predetermined time, a current value is measured byelectrode reaction to determine the quantity of the component in theplasma.

FIG. 4 shows an example of how the reaction layers are arranged in thevicinity of the electrode system in the sample solution supply pathway12′. On the electrode system on the insulating base plate 1, are formeda hydrophilic polymer layer 28 containing sodium salt of carboxymethylcellulose (hereinafter simply referred to as “CMC”) and a reaction layer29 containing a reaction reagent such as an electron mediator. In thesample solution supply pathway 12′ formed by combining the cover 13 andthe spacer 7, a reaction layer 29′ containing oxidoreductase is formedon the surface of the cover 13 exposed to the sample solution supplypathway 12′.

As shown in FIGS. 1 to 4, in the sample solution supply pathway 12′, adistance in a direction vertical to the liquid flow is made smaller thanthe thickness of the primary side portion of the filter 6, whereas aportion of 1 mm from the secondary side end of the filter 6 iscompressed to be positioned in the vicinity of the connecting part 11 ofthe sample solution supply pathway 12′.

The compressed portion of the filter 6 preferably occupied about 1 mmfrom the filter tip on the secondary side with respect to suction powerof a sensor sized as described in the following Example of the presentinvention. The secondary side portion of the filter 6 was preferablycompressed to such a degree that the secondary side portion becomesabout ¼ to ⅓ of the primary side portion.

Although it is difficult to express the suction power of the sensor by anumeric value, favorable measurement result (flow rate) was obtainedwhen the spacer 7 is 100 μm in thickness and the filter was compressedto a thickness of 370 μm. The flow rate was low where the filterthickness was 310 μm or less.

Thus, with the sample solution supply pathway 12′ formed smaller thanthe primary side portion of the filter 6 in unit area in cross section,plasma from which hemocytes are removed by the filter 6 is suckedrapidly into the sample solution supply pathway 12′ due to capillarity.

In general, the reaction layer is easy to dissolve in one portion andhard to dissolve in other portion. The easy-to-dissolve portion liesalong the edge of the sample solution supply part 12′, i.e., along thewall surface of the slit 12 of the spacer 7. The hard-to-dissolveportion is a center portion of the reaction layer in the liquid flowdirection. Since the sample solution having passed the filter 6 flowsalong the slit 12 by priority, the sample solution may fill the airaperture in some cases before the center portion of the reaction layerdissolves completely. With the secondary side portion of the filter 6shaped such that a center thereof is projected inside of the samplesolution supply pathway 12′ as compared with the right and left, thesample solution flows along the center portion of the sample solutionsupply pathway 12′ by priority. Thereby, the plasma can rapidly be flowninto the sensor without leaving bubbles in the center portion of thesample solution supply pathway 12′.

In measurement, a fingertip stung to bleed is placed on the samplesolution supply part 30 to supply blood to the filter 6. The bloodpermeates into the filter 6 from the end face and the top face on theprimary side. At this time, with the existence of the third pressingpart c serving as a partition, the blood does not travel on the surfaceof the filter 6 by priority to flow directly into the sample solutionsupply pathway 12′. Further, since the third and first pressing parts cand a do not agree in position as viewed in a projection on a planeidentical to the insulating base plate 1, the expansion of the filter 6is not inhibited and the possibility of destroying the hemocytes iseliminated.

The electrode system is preferably made of noble metal electrodes. Ifprinted electrodes formed by screen printing are used, accuracy indetermining the electrode area becomes poor because the preferable widthof the sample solution supply pathway 12′ is 1.5 mm or less. On theother hand, the noble metal electrodes allow trimming with laser of 0.1mm width, which is highly accurate in determining the electrode area.

Hereinafter, an example of the present invention is described, but theinvention is not limited thereto.

EXAMPLE

A cholesterol sensor configured as shown in FIGS. 1, 2 and 4 wasfabricated in the following manner. An electron mediator was containedin a reaction layer 29 and cholesterol oxidase, cholesterol esterase anda surfactant were contained in a reaction layer 30.

First, 5 μl of 0.5 wt % CMC aqueous solution was dropped onto theelectrode system of the insulating base plate 1 and dried in a warm-airdryer at 50° C. for 10 minutes to form a hydrophilic polymer layer 28.

Then, 4 μl of potassium ferricyanide aqueous solution (corresponding to70 mM of potassium ferricyanide) was dropped onto the hydrophilicpolymer layer 28 and dried in the warm-air drier at 50° C. for 10minutes to form a reaction layer 29 containing potassium ferricyanide.Further, to a solution dissolved therein cholesterol oxidase derivedfrom Nocardia (EC1.1.3.6: ChOD) and cholesterol esterase derived fromPseudomonas (EC.3.1.1.13: ChE), polyoxyethylene (10) octyl phenyl ether(Triton X-100) was added as a surfactant.

The resulting mixture solution was dropped in an amount of 0.4 μl onto aportion of the cover 13 exposed to the sample supply solution pathway12′, preliminarily frozen with liquid nitrogen at −196° C., and thendried using a freeze dryer for 2 hours to form a reaction layer 30containing 450 U/ml of cholesterol oxidase, 1125 U/ml of cholesterolesterase and 2 wt % of a surfactant.

Glass fiber filter paper of about 300 to 400 μm thick was stamped intothe form of an isosceles triangle having a bottom of 3 mm and a heightof 5 mm and a tip thereof on the secondary side was rounded to obtain afilter 6. The filter 6 of an almost isosceles triangle shape wasdisposed between the insulating base plate 1 and the joint base plate A.

Thereafter, the member obtained by disposing the filter 6 between theinsulating base plate 1 and the joint base plate A was bonded to thejoint base plate B obtained by integrating the spacer 18 and the cover22, thereby forming a cholesterol sensor configured as shown in FIGS. 1,2 and 4.

In this sensor, 10 μl of whole blood sample solutions varied inconcentration were added to the sample solution supply part 30. Threeminutes later, a pulse voltage of +0.2V with reference to the counterelectrode was applied to the measuring electrode, i.e., in the anodedirection, and then 5 seconds later, a current value between themeasuring electrode and the counter electrode was measured. The resultsare shown in FIG. 5, which is a graph illustrating a relationshipbetween the cholesterol concentration in the whole blood and the currentvalue.

As apparent from FIG. 5, the sensor of the present invention givesfavorable linearity between the cholesterol concentration and thecurrent value.

INDUSTRIAL APPLICABILITY

According to the present invention, there is provided an improvedbiosensor in which plasma stops at an air aperture regardless of theviscosity of the plasma in a sample solution and the plasma volume inwhole blood. Thus, the present invention provides a cholesterol sensorof high accuracy and excellent response aimed at measurement of thewhole blood.

1. A biosensor comprising an insulating base plate, an electrode systemincluding a measuring electrode and a counter electrode provided on saidbase plate, a cover for covering said insulating base plate, at leastone reaction layer containing oxidoreductase and/or an electronmediator, a sample solution supply pathway which is formed by saidinsulating base plate and said cover and includes said electrode systemand said reaction layer, a first air aperture provided in said cover insaid sample solution supply pathway, a sample solution supply part, anda filter provided between said sample solution supply pathway and saidsample solution supply part to filter hemocytes, said biosensor beingcapable of sucking plasma with hemocytes therein filtered with saidfilter into said sample solution supply pathway due to capillarity,characterized in that a second air aperture is provided in saidinsulating base plate in said sample solution supply pathway.
 2. Thebiosensor in accordance with claim 1, characterized in that said firstair aperture and said second air aperture are communicated.
 3. Thebiosensor of claim 2, wherein said second air aperture is provided at aposition contacting said measuring electrode.